1. Field of the Invention
The instant invention pertains to a kinetic method for directly determining total bilirubin.
2. Description of the Prior Art
The development of methodologies for the determination of bilirubin in biological fluids has been documented (1, 13). In general, endpoint techniques employing various accelerators and stabilized or unstabilized diazotized aromatic amines have been employed to measure total bilirubin (2-12 and 14-21).
Recently, Rizi et al. (22) disclosed a kinetic method for measuring conjugated bilirubin but still employed an endpoint technique to measure total bilirubin.
The reaction kinetics of conjugated and unconjugated bilirubin have been examined (23-25). More particularly, Landis et al. (24) discuss the kinetics of the reactions of unconjugated and conjugated bilirubin with p-diazobenesulfonic acid. Landis et al. (24) confirm that both the unconjugated bilirubin reaction and the conjugated bilirubin reaction each proceed in the following two steps, respectively: ##EQU1## wherein B.sub.uc is unconjugated bilirubin; B.sub.c is conjugated bilirubin; D is p-diazobenzenesulfonic acid; K.sub.luc, K.sub.2uc, K.sub.3c, and K.sub.4c are the rate constants for reactions I through IV, respectively; A.sub.1 and A.sub.2 are azobilirubin isomers; and HPC is hydroxypyromethene carbinol. In the presence of an excess of p-diazobenzenesulfonic acid and in the absence of sulfanilic acid, Landis et al. (24) report that the reaction for either unconjugated or conjugated bilirubin proceeds in two successive first-order steps wherein K.sub.luc &gt;&gt;K.sub.2uc and K.sub.3c &gt;&gt;K.sub.4c. Landis et al. (24) also report that in the presence of excess sulfanilic acid, K.sub.2uc approaches K.sub.luc and K.sub.4c approaches K.sub.3c and that the system exhibits complex kinetic behavior and is difficult to treat quantitatively.
In addition to the above, Landis et al. (24) also state that the reactions have half-lives in the range from 0.002 to 0.4 seconds (depending upon the reaction conditions) and, therefore, these authors employed a stopped-flow mixing system to achieve the needed mixing times.
In the second Landis et al. article (25), the authors describe a kinetic method that permits simultaneous determination of both unconjugated bilirubin and conjugated bilirubin in a single step in the same reaction mixture. This method makes use of the fact that at certain pH values, K.sub.luc differs significantly from K.sub.3c, and this difference in K.sub.luc and K.sub.3c permits the two species to be resolved quantitatively by use of kinetic data.
Landis et al. (25) reason that the kinetic method for simultaneous determination of conjugated and unconjugated bilirubin would be more attractive in the immediate future if K.sub.luc and K.sub.3c were slow enough to be handled with conventional instrumentation. Caffeine concentration and pH are noted by Landis et al. (25) as being variables that might be manipulated to slow the reaction. However, Landis et al. (25) state that, unfortunately, the caffeine concentration used in their work was near its upper solubility limit. Landis et al. (25) conclude by pointing out that significant additional work is needed to evaluate the possibility that a pH below 5 could result in reactions slow enough to be monitored with conventional mixing systems and fast enough to give reasonable analysis times.
Despite the fact that reaction kinetics of conjugated and unconjugated bilirubin have been examined, no one has yet suggested a kinetic method for directly determining total bilirubin. This fact is significant in view of the extensive research undertaken in the area of bilirubin assays (26-37).
As known to those skilled in the art, kinetic methods for performing an assay have very significant advantages. These advantages include, but are not limited to, rapidity of the assay; freedom from interfering substances in the assay medium; and the ability to make accurate determinations without running blank reactions.